The overall objective of the proposed research is to determine in molecular detail several of the fundamental interrelationships of structure, function and reactivity of [4Fe4S] proteins. Differences in their capabilities are determined by: a) the type of ligands that coordinate the Fe of the cluster, b) the exchange of an Fe atom by a different metal and c) the loss of an Fe atom. In particular we have chosen to examine C pasteurianum ferrodoxin (Fd) since it has two [4Fe-4S] clusters, has a relatively low molecular weight of 6300, is highly homologous to Peptococcus aerogenes Fd whose X-ray structure has been determined, and has excellent spectroscopic properties for following cluster integrity and environment. We propose to use proven solid phase peptide synthetic methods to prow a new series of apo-Fds with site specific amino acid (and/or stable isotopic) substitutions and reconstitute the apoproteins to the holo-Fds. Solid phase methods provide a unique method of preparing single site derivatives that are virtually unobtainable through molecular biological methods. Several questions addressed: 1. Can we determine the absolute stereospecific assignments for the 16 Cys beta-CH2 protons through the use of 2H and 13 C-cysteine substitutions at specific Cys positions? 2. What is the detailed mechanism(s) of the folding and unfolding of Fd? We propose to examine the formation of holoprotein from apoprotein (native and synthetic) in the presence of water soluble clusters e.g. [4Fe-4S](SCH2CH2CO2-)4. 3. What are the structural requirements for thermal stability of iron-sulfur proteins and how is that stability reflected in the electron transfer capabilities of each Fd? 4. What are the specific structural requirements that enable a specific cluster Fe atom to either exchange with exogenous metal, to be lost. to form a [3Fe-4S]. Substitution of specific cysteines of C.p. Fd with new amino acids will provide an opportunity to explore the mechanism and dynamics of these processes. 5. Are the clusters truly identical in their chemical reactivity? Recent work in our lab indicates that the cluster differ in their reactivity; we propose to determine why one is more reactive than the other. 6. Stereospecific resonance Raman, spectroscopic assignments will be made with specific CyS 13C and 2H derivatives. 7. The influence of specific substitutions on redox and electron transfer capabilities will be examined.